Tissue regenerative composition, method of making, and method of use thereof

ABSTRACT

A matrix, including epithelial basement membrane, for inducing repair of mammalian tissue defects and in vitro cell propagation derived from epithelial tissues of a warm-blooded vertebrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 13/228,795 filed Sep. 9, 2011 and entitled TISSUE REGENERATIVECOMPOSITION, METHOD OF MAKING, AND METHOD OF USE THEREOF, which in turnis a continuation of U.S. patent application Ser. No. 10/337,152 filedJan. 6, 2003, which in turn is a continuation of U.S. patent applicationSer. No. 09/691,345 filed Oct. 18, 2000, now granted U.S. Pat. No.6,576,265, which issued Jun. 10, 2003 and claims priority to and thebenefit of U.S. provisional patent application Ser. No. 60/171,733,filed Dec. 22, 1999, the content of each of which is incorporated byreference herein in its entirety for all purposes.

TECHNICAL FIELD

This invention relates to devitalized acellular tissue regenerationcompositions, methods of making, and methods of use.

BACKGROUND OF THE INVENTION

Submucosal tissues of warm-blooded vertebrates are useful in tissuegrafting materials.

For example, submucosal tissue graft compositions derived from smallintestine have been described in U.S. Pat. No. 4,902,508 (hereinafterthe '508 patent) and U.S. Pat. No. 4,956,178 (hereinafter the '178patent), and submucosal tissue graft compositions derived from urinarybladder have been described in U.S. Pat. No. 5,554,389 (hereinafter the'389 patent).

All of these compositions consist essentially of the same tissue layersand are prepared by the same method, the difference being that thestarting material is small intestine on the one hand and urinary bladderon the other. The procedure detailed in the '508 patent, incorporated byreference in the '389 patent and the procedure detailed in the '178patent, includes mechanical abrading steps to remove the inner layers ofthe tissue, including at least the luminal portion of the tunica mucosaof the intestine or bladder, i.e., the lamina epithelialis mucosa(epithelium) and lamina propria, as detailed in the '178 patent.Abrasion, peeling, or scraping the mucosa delaminates the epithelialcells and their associated basement membrane, and most of the laminapropria, at least to the level of a layer of organized dense connectivetissue, the stratum compactum. Thus, the tissue graft materialpreviously recognized as soft tissue replacement material is devoid ofepithelial basement membrane and consists of the submucosa and stratumcompactum.

The epithelial basement membrane is a thin sheet of extracellularmaterial contiguous with the basilar aspect of epithelial cells. Sheetsof aggregated epithelial cells of similar type form an epithelium.Epithelial cells and their associated epithelial basement membrane arepositioned on the luminal portion of the tunica mucosa and constitutethe internal surface of tubular and hollow organs and tissues of thebody. Epithelial cells and their associated epithelial basement membraneare also positioned on the external surface of the body, i.e., skin.Examples of a typical epithelium having a basement membrane include, butare not limited to the following: the epithelium of the skin, intestine,urinary bladder, esophagus, stomach, cornea, and liver.

Epithelial cells are positioned on the luminal or superficial side ofthe epithelial basement membrane, opposite to connective tissues.Connective tissues, the submucosa, for example, are positioned on theabluminal or deep side of the basement membrane. Examples of connectivetissues that are positioned on the abluminal side of the epithelialbasement membrane are the submucosa of the intestine and urinarybladder, and the dermis and subcutaneous tissues of the skin.

SUMMARY OF THE INVENTION

The present invention provides devitalized tissue regenerativecompositions comprising an epithelial basement membrane as part of amatrix or scaffold for tissue repair or regeneration. The inclusion ofthe epithelial basement membrane in devitalized mammalian tissueregenerative compositions results in improved in vivo endogenous cellpropagation and tissue restoration as compared to submucosal matricesdescribed above which do not include an epithelial basement membrane.For the purposes of this invention, devitalized means acellular orsubstantially acellular. For the purposes of this invention, epithelialbasement membrane means at least a portion of the intact epithelialbasement membrane.

According to the invention, a preferred devitalized matrix for mammaliantissue repair or regeneration comprises at least a portion of amammalian epithelial basement membrane, preferably the entire epithelialbasement membrane, and the tunica propria that is immediately subjacentto the basement membrane. Devitalized matrices of the invention restoreor replace diseased, defective, or missing tissue when placed in contactwith host tissue. In a preferred embodiment, the invention comprises adevitalized matrix that is custom-shaped to conform to the diseased ordefective tissue. In a particular embodiment, the matrix comprises asheet of matrix derived from the urinary bladder, the intestine, or anyother mammalian epithelial tissue. In another embodiment, the matrix isinjectable by means of being transformed into a fine particulate,emulsion, gel or extract. A matrix of the invention may act as a carrierfor a pharmaceutical agent. A preferred application of the matrix of theinvention is the repair or restoration of cardiac tissue. In particular,a matrix or composition of the invention is useful to restore or replaceat least a portion of a cardiac valve, the interatrial septum, theinterventricular septum, or the myocardium. For the purposes of thisinvention, matrix and composition are interchangeable terms.

In one embodiment, the invention features a devitalized compositioncomprising epithelial basement membrane and tunica propria immediatelysubjacent to the basement membrane. The epithelial basement membrane andtunica propria immediately subjacent to the basement membrane aredelaminated from cells of a mammalian epithelium and abluminal portionsof the tunica propria. Mammalian epithelial tissue used in this aspectof the invention is preferably derived from urinary bladder, intestine,or any other mammalian epithelial tissue. Further embodiments feature acomposition shaped to conform to a diseased or defective cardiac valvesuch as at least a portion of a pulmonic valve, aortic valve, right orleft atrioventricular valve, or the myocardium.

In still another embodiment, the invention features a compositioncomprising epithelial basement membrane, tunica propria, and submucosa.The epithelial basement membrane and tunica propria are delaminated fromthe cells of an epithelium and from the tunica muscularis of a mammalianepithelial tissue.

In yet another embodiment, the invention features a compositioncomprising epithelial basement membrane, tunica propria, and smoothmuscle cells of the tunica muscularis, all delaminated from epithelialcells of a mammalian epithelium.

The composition, according to the invention, is not limited to merelythe embodiments enclosed. Rather, the composition, according to theinvention, comprises one or more layers of an epithelial tissue incombination with at least a portion, preferably the entire, intactepithelial basement membrane.

In another aspect, the invention provides methods for inducingrestoration or repair of diseased or defective cardiac tissue. Apreferred method of the invention comprises the step of contacting ahost tissue with a devitalized matrix derived from a mammal. Thedevitalized matrix comprises at least a portion of an epithelialbasement membrane and tunica propria immediately subjacent to thebasement membrane. In preferred embodiments, methods of the inventioncomprise inducing endogenous epithelial repair using tissue regenerativecompositions of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are not to scale and emphasis instead is generally beingplaced upon illustrating the principles of the invention.

FIG. 1A is a cross-sectional view of the wall of the intestine.

FIG. 1B is a cross-sectional view of the wall of the urinary bladder.

DETAILED DESCRIPTION OF THE INVENTION

A devitalized tissue regenerative composition in accordance with thepresent invention comprises epithelial basement membrane or at least aportion of the epithelial basement membrane and at least the subjacentportion of the tunica propria harvested from a mammalian epithelialtissue. Preferred epithelial tissues for use in the invention include,but are not limited to, urinary bladder and other tissues of theuro-genital tract, small intestine, esophagus and other tissues of thegastrointestinal tract, skin, liver, and arteries such as the aorta andother tissues of the cardiovascular system. In a preferred embodiment,the invention provides a tissue graft composition comprising at least aportion of the epithelial basement membrane and subjacent tunicapropria, separated from the luminal epithelial cells, the abluminaladventitial, serosal, and smooth muscle layers and the submucosal tissuelayers Tissue separation or delamination techniques, according to theinvention, provide a layer of devitalized extracellular matrix materialincluding epithelial basement membrane or at least a portion of theepithelial basement membrane essentially free of cells. Any remainingcellular elements are then removed by further processing steps such asrinsing in hypotonic saline, peracetic acid or sterile water.

Accordingly, referring to FIGS. 1A and 1B, a preferred embodiment of theinvention comprises epithelial basement membrane B and the biotropicconnective tissue known as the tunica propria C that is immediatelysubjacent to and positioned on the abluminal side of the epithelialbasement membrane B of the intestine illustrated in FIG. 1A, or theurinary bladder illustrated in FIG. 1B or any other epithelial tissue.This embodiment of the invention features the epithelial basementmembrane B and portions of the tunica propria C adjacent to theepithelial basement membrane B. The epithelial basement membrane B andtunica propria C are delaminated from the epithelial cells A, thesubmucosa D, the tunica muscularis E, and the serosa F. Thus, in thisembodiment of the invention, the portions of the tunica mucosa Hadjacent the lumen L, i.e., the luminal portions of the tunica mucosa,form a preferred tissue matrix composition.

In another preferred embodiment, again referring to FIGS. 1A and 1B, acomposition of the invention comprises epithelial basement membrane B,tunica propria C, and the tunica submucosa D. The epithelial basementmembrane B, tunica propria C, and the tunica submucosa D are delaminatedfrom the epithelial cells A, tunica muscularis E, and tunica serosa F.In this embodiment, the portions of the tunica mucosa HI that includethe epithelial basement membrane, and the tunica submucosa, form apreferred tissue matrix composition.

In yet another composition, a preferred embodiment of the inventioncomprises the epithelial basement membrane B, the tunica propria C thatlie adjacent the epithelial basement membrane B, the tunica submucosa Dand at least a portion of the tunica muscularis E.

Sources of Epithelial Tissue

Material for tissue regeneration compositions of the invention istypically prepared from tissue harvested from animals raised for meatproduction, including but not limited to, pigs, cattle and sheep. Otherwarm-blooded vertebrates are also useful as a source of tissue, but thegreater availability of such tissues from animals used for meatproduction makes such tissue preferable. Thus, there are inexpensivecommercial sources of tissue for use in preparation of the tissuecompositions in accordance with the present invention. There may bespecially bred or genetically engineered strains of certain species thatare used as a tissue source. For example, pigs that are geneticallyengineered to be free of the galacatosyl, alpha 1,3 galactose (GALepitope) may be used as the source of tissues for production of thecomposition. Alternatively, herds of pigs that are raised to be free ofspecific pathogens may be used as the source of tissues. Mammaliantissue used as the source of tissue for production of the composition ofthe invention may be harvested from an animal of any age group,including embryonic tissues, market weight, gender or stage of sexualmaturity.

Tissue Sources of Epithelial Basement Membrane

Urinary Bladder

A preferred source of epithelial basement membrane is the urinarybladder illustrated in FIG. 1B of a warm-blooded vertebrate such as apig. Superior biologic tissue remodeling properties are derived fromepithelial basement membrane components that support and promote cellgrowth without invasion, and the subjacent tunica propria matrixmaterial that allows and promotes endogenous cellular adhesion,invasion, growth and differentiation. The matrix, referred tohereinafter as urinary bladder matrix (UBM), includes the basementmembrane B of the urinary bladder epithelium and the subjacent tunicapropria C. In this embodiment, epithelial basement membrane B and thesubjacent tunica propria C are delaminated from the epithelial cells Aand the extracellular matrix of the tunica submucosa D, the tunicamuscularis E, and the tunica serosa F. UBM is harvested from anywarm-blooded vertebrate but is most preferably harvested from pigs. UBMis used as a bioscaffold for the repair or restoration of body tissuesand organs such as musculoskeletal and cardiovascular structures,dermatologic and gastrointestinal tissues, urogenital, and reproductivetissues, neurologic tissues, liver, kidney, and head and neck tissues.

A preferred UBM tissue regenerative composition comprises epithelialbasement membrane, preferably urinary bladder basement membrane, and thebiotropic molecular structure that lies immediately subjacent theepithelial basement membrane from the urinary bladder tissue of warmblooded vertebrates. In this embodiment, epithelial basement membrane isdelaminated from the luminal epithelial cells, abluminal adventitial,serosal, and smooth muscle tissues, and submucosal tissues. Tissue graftcompositions of the invention have remarkably superior tissue growthcharacteristics as compared to previously described submucosal tissuegraft compositions implanted or injected into a vertebrate host to causethe repair or replacement of damaged, missing, or defective tissues ororgans.

Methods of the present invention avoid complete loss of the epithelialbasement membrane and result in a tissue regenerative composition thatincludes at least a portion of the epithelial basement membrane. In apreferred embodiment, the epithelial basement membrane as determined byconventional histochemical or immunohistochemical techniques and light,or electron microscopy, is largely intact. The resulting devitalizedmaterial obtained by methods of the present invention is in contrast tomethods for making tissue graft compositions derived from smallintestine and urinary bladder as described in the '508 and '389 patentswhich result in a graft material including submucosa exclusive of theepithelial basement membrane. Steps in preparation of UBM from urinarybladder tissue differ from previously described steps for preparation ofsubmucosal tissue graft composition described in the '508 patent and the'389 patent. In the methods for the preparation of the submucosal tissuegraft composition described in the '508 and '389 patents, the mucosa ismechanically removed by abrasion.

According to the present invention, UBM is prepared by removing theurinary bladder tissue from a warm-blooded vertebrate, for example, apig, and delaminating the tissue by first soaking the tissue in adeepithelializing solution, for example, hypertonic saline, mostpreferably 1.0 N saline, for periods of time ranging from 10 minutes to4 hours. Exposure to hypertonic saline solution effectively removes theepithelial cells from the underlying basement membrane. The tissueremaining after the initial delamination procedure includes epithelialbasement membrane and the tissue layers abluminal to the epithelialbasement membrane. This tissue is next subjected to further treatment toremove the majority of abluminal tissues but not the epithelial basementmembrane. The outer serosal, adventitial, smooth muscle tissues,submucosa and abluminal portion of the tunica propria are removed fromthe remaining deepithelialized tissue by mechanical abrasion or by acombination of enzymatic treatment, hydration, and abrasion. Mechanicalremoval of these tissues is accomplished by removal of mesenterictissues with, for example, Adson-Brown forceps and Metzenbaum scissorsand wiping away the tunica muscularis and abluminal tunica propria usinga longitudinal wiping motion with a scalpel handle or other rigid objectwrapped in moistened gauze. After these tissues are removed, theresulting tissue scaffold consists of epithelial basement membrane andsubjacent tunica propria. This tissue differs from previously knowntissue compositions derived from animal epithelial tissues by theinclusion of a largely intact epithelial basement membrane in thepresent invention. The tissues may be further processed by rinsing inhypertonic saline, peracetic acid or sterile water. Other methods forremoving tissue layers, a microtome, for example, may also be used toobtain the tissue composition of the invention.

The method for preparation of tissue regenerative compositions accordingto the invention is not limited to the use of urinary bladder tissue asa starting material. The method according to the invention is alsoapplicable to other starting tissues, for example, skin, esophagus,stomach, and intestinal tissues.

After preparing UBM according to the method of the invention, theresulting tissue scaffold consists of an approximately 10-120 micrometerthick material that consists primarily (i.e., greater than 90%) ofextracellular matrix (ECM) including the epithelial basement membrane.This material may or may not retain some of the cellular elements thatcomprised the original tissue such as capillary endothelial cells orfibrocytes. These cellular elements are removed by subsequent exposureto peracetic acid as part of the disinfection of the biomaterial. Thematerial differs in its histologic appearance and its architecture fromthe submucosal tissue graft compositions because of the smoothepithelial basement membrane that demarks the luminal surface and thedense, partially organized collagenous ECM that demarks the abluminalsurface. The ECM material stains pink with H&E stain and blue withMasson's trichrome stain.

Skin, Esophagus

Similarly, steps used in preparation of tissue regenerative compositionsfrom other epithelial organs having tissue layers similar to urinarybladder, such as skin, or esophagus, parallel the steps described abovefor preparing UBM. Like the urinary bladder matrix, the materialremaining after removal of the epithelial cells, tunica serosa, tunicamuscularis and abluminal tunica propria, includes at least a portion ofthe epithelial basement membrane, and the adjacent tunica propria.

Small Intestine

A tissue regenerative composition of the invention is also derived fromepithelial tissues of the gastrointestinal tract, such as the smallintestine. Steps in preparation of a tissue regenerative compositionthat includes at least a portion of the epithelial basement membrane ofthe small intestine and subjacent tunica propria, termed SIM, aresimilar to the steps described above for the formation of UBM. 1.0Nsaline may be used to remove the intestinal epithelial cells from theepithelial basement membrane. An alternate method for removingepithelial cells is to soak the epithelial tissue in a detergent such asa non-ionic detergent, for example, Triton X-100, at concentrations from0.025 to 1%, for 5 minutes to several hours.

In one embodiment, the delaminated tissue regenerative compositionderived from an epithelial tissue is stored either in a frozen hydratedstate or is air dried at room temperature, then stored. Alternatively,the tissue regenerative composition is lyophilized and stored in adehydrated state at either room temperature or frozen. In yet anotherembodiment, the tissue regenerative composition can be minced andfluidized by digesting the material in proteases, for example pepsin ortrypsin, for periods of time sufficient to solubilize the tissue andform a substantially homogeneous solution. The viscosity of thesolubilized material can be varied by adjusting the pH to create a gel,gel-sol, or completely liquid state. The preparation of fluidizedintestinal submucosa, for example, is described in U.S. Pat. No.5,275,826, expressly incorporated herein by reference.

In still another embodiment, the present invention contemplates the useof powder forms of the tissue regenerative composition. In oneembodiment, a powder form of tissue regenerative composition is createdby mincing or crushing the delaminated material to produce particlesranging in size from 0.005 mm² to 2.0 mm². The material, delaminatedfrom unwanted tissue layers, is frozen for example, in liquid nitrogen,to perform the crushing procedure. Alternatively, the material isdehydrated to perform the crushing procedure. The crushed form of thematerial is then lyophilized to form a substantially anhydrousparticulate tissue regenerative composition.

Tissue compositions of the present invention are suitable for manysurgical and nonsurgical applications for the purpose of inducingreconstructive wound healing and tissue restoration. For example, theyare used to replace damaged, diseased, or missing heart valves,arteries, veins, urinary bladder, liver, portions of thegastrointestinal tract, or they can be used as templates for repair orreplacement of head and neck structures. The material, in any of anumber of its solid or fluidized forms, can be used as a scaffold fordermal or epidermal repair, injected into various body sphincters suchas urinary sphincter or esophageal or gastric sphincters, folded into atube or partial tube as a conduit for the restoration of nervous tissueor extruded or molded into any shape suitable for its application as atissue regenerative composition. The tissue regenerative composition ofthe invention can be sutured into place in its solid sheet form, placedin wounds or body locations in a gel form, or injected in its liquid orparticulate form. Tissue compositions of the present invention inducegrowth of endogenous tissues including epithelial and connective tissueswhen target tissues in vivo are placed in contact with mammalianderived, devitalized tissue compositions comprising at least a portionof an epithelial basement membrane.

Urinary Bladder Matrix (UBM)

UBM compositions comprise at least type I and type IV collagen,glycosaminoglycans, including hyaluronic acid, chondroitin sulfate A andB, heparin and heparin sulfate. In addition, one or more of basicfibroblast growth factor, vascular endothelial cell growth factor andTGF-beta are present in UBM.

The physical properties of UBM have been partially characterized. UBMhas a uniaxial strength of approximately 0.1-2.0 pound per 1.0 cm widestrip (measured with a material testing system machine via AmericanStandards for Testing Materials pulling at 1 inch/minute). The sutureretention strength of the material is approximately 1.0-4.0 Newtons (N)per sheet layer, specifically, 4-18 N for a 4 layer matrix and 30 N-120N for a 30 layer matrix. The ball burst test failure force isapproximately 4-10 pounds for each layer, specifically, 32-80 N for 8layers, 16-40 N for 4 layers, and 36-120 N for 12 layers.

The porosity index is defined as the amount of water that flows througha material per cm²/minute at 120 mmHg pressure. Water porosity differsfrom one side of UBM to the other depending on the direction of flow.Water flows from the epithelial basement membrane to the abluminal sideat approximately 20% the rate of water flow from the abluminal side tothe epithelial basement membrane side of the matrix. UBM also hasviscoelastic properties.

UBM can be sterilized by any of a number of standard methods withoutloss of its ability to induce endogenous tissue growth. For example, thematerial, after rinsing in saline and peracetic acid at 0.05% to 1.0%,can be sterilized by ethylene oxide treatment, gamma irradiationtreatment (0.5 to 2.5 mRad), gas plasma sterilization, or e-beamtreatment. The material can also be sterilized by treatment withglutaraldehyde that causes cross linking of the protein material, butthis treatment substantially alters the material such that it is slowlyresorbed or not resorbed at all and incites a different type of hostremodeling which more closely resembles scar tissue formation orencapsulation rather than constructive remodeling. Cross-linking of theprotein material can also be induced with carbodiimide or dehydrothermalor photooxidation methods.

The following examples will serve to better demonstrate the successfulpractice of the present invention.

EXEMPLIFICATION

As exemplification of the utility of methods and compositions of theinvention, UBM is applied to heart valve defects. As will be appreciatedby those of ordinary skill in the art, methods and compositionsdisclosed herein are applicable to other tissue regenerativecompositions derived from sources of epithelial tissue other than theurinary bladder, from mammalian sources other than pigs, and to tissuedefects other than heart valve. Moreover, tissue regenerativecomposition of the invention can be applied in a form other than a sheetor multilayer sheet of material, for example, UBM may be applied as anextract, in gel form, powdered form, tubular form, sheet form, or asstrips, cords or struts or mixed with other pharmaceutical agents, forexample, growth factors and gene products. UBM may be extruded or moldedin or on a form to fit a particular application in the body. Thepreparation of fluidized forms of tissue is described in U.S. Pat. No.5,275,826, the disclosure of which is incorporated herein by reference,and the preparation of solid sheets and strips of tissue is described inU.S. Pat. No. 5,711,969, the disclosure of which is incorporated hereinby reference.

Application 1: Cardiac Tissue Repair

One embodiment, according to the invention, is a tissue regenerativecomposition for repair or replacement of cardiac tissues. Cardiactissues include, but are not limited to, diseased, damaged, or missingheart tissue including myocardium, epicardium, endocardium, pericardium,interatrial and interventricular septum and all heart valves andassociated valve leaflets including pulmonic valve, aortic valve, rightatrioventricular valve and left atrioventricular valve and portions ofadjacent vessels of the heart including pulmonary artery, pulmonaryvein, aorta, inferior vena cava, and superior vena cava.

In this embodiment of the invention disclosed herein, UBM was preparedfrom porcine urinary bladder as described above, and used as autogenicand xenogenic anterior heart valve replacement leaflet of the pulmonicvalve in five pigs and three dogs.

UBM, configured as a single sheet of material or as double thicknessmaterial, was cut with scissors or a scalpel at the time of surgery tofit the pulmonic valve anterior leaflet. UBM was sutured directly to theannulus at the base of the valve. In the single sheet embodiment, theepithelial basement membrane side of UBM was positioned on the rightventricular luminal side of the replacement valve leaflet and sutureddirectly to the annulus of the pulmonic valve. In a double thicknessembodiment of UBM, UBM was folded so that the epithelial basementmembrane was positioned on both surfaces, i.e., ventricular and arterialsurfaces, of the replacement pulmonic valve leaflet, and sutureddirectly to the annulus of the pulmonic valve.

The pulmonic valves of experimental dogs and pigs were examined 6 and 12weeks after valve replacement. One dog was examined at 5 months aftervalve leaflet replacement. Standard tissue fixation andhistopathological techniques were used to examine the harvested valveleaflets.

At six weeks post valve leaflet replacement, epithelialization of thereplacement valve leaflet was present over the entire valve leafletsurface. Cells migrating over the valve leaflet surface stained positiveby immunofluorescent staining for von Willebrand factor indicating thatthese cells were of endothelial origin. In some valve leaflets some ofthe endothelial cells had features of early progenitor cells.Neovascularization, endothelial cell infiltration, and deposition ofextracellular matrix were observed originating from the host tissue atthe annulus of the pulmonic valve and extending into the replacementvalve leaflet.

At twelve weeks and at five months post valve leaflet replacement, noneof the original UBM tissue composition was recognizable and restorationof the valve leaflet was complete. Unexpected findings at all timepoints examined included lack of endothelial invasion into thereplacement valve leaflet, lack of thrombosis, and lack of calcificationor cell-mediated rejection of the replacement valve leaflet. Moreover,the shape of the replacement valve leaflet was unchanged from the shapeof the original valve leaflet, at all time points examined.

Ultrasound studies of the pulmonic valve in pigs at 8, 12, 16 and 20weeks after valve replacement demonstrated a competent valve.

In another embodiment of this aspect of the invention, fluidized,powderized, or pulverized forms of UBM are applied to or injected intoor adjacent to diseased or defective cardiac tissue to promoteendogenous tissue repair. For example, fluidized UBM is injected into oradjacent to a congenital interventricular septal defect, congenitalinteratrial septal defect, or into the lumen of a patent ductusarteriosus to promote endogenous growth of tissue in these areas.

Application of UBM to cardiac tissues is accomplished by the applicationof several different surgical approaches. For example, a minimalinvasive procedure is used to approach the cardiac surgical site withthe aid of a laproscope. Alternatively, a thoracotomy is performed. UBMis brought to the surgical site in any of its prepared forms such as asheet, loop, strip or as an injectable, powered, or pulverized form.Sheets or strips of UBM are custom-fit for the particular cardiacapplication before or during the surgical procedure. Sheets or strips ofUBM are secured adjacent to or in the defective or diseased cardiactissue with sutures, staples, tissue glue, or any other means known toone skilled in the art.

Application 2: Matrix for In Vitro Cell Proliferation

Human microvascular endothelial cells (HMVEC) form endothelium, a singlelayer of cells organized on a basement membrane in vivo in a manner thatmimics epithelium. Studies were conducted in vitro using isolated HMVECplated on (i) the epithelial basement membrane side of a sheet of UBM,(ii) the abluminal surface of UBM, (iii) small intestine submucosatissue graft composition (SIS) prepared according to the methodsdisclosed in the '508 and '178 patents, and (iv) urinary bladdersubmucosa tissue composition (UBS) prepared according the methoddisclosed in the '389 patent.

HMVEC grew into the matrix and did not form a confluent cell layerfollowing three days' growth when plated on the surface of SIS and UBSregardless of whether HMVEC were plated on the luminal or abluminalsurface of SIS or UBS.

HMVEC plated on the abluminal surface of UBM grew into the matrix,proliferated and differentiated into mature endothelial cells. LikeHMVEC plated on the abluminal surface of SIS and UBS, a confluent layerof HMVEC was not formed on the abluminal surface of UBM following threedays' growth.

In contrast to other previously known tissue regenerative compositionssuch as SIS and UBS in these studies, HMVEC plated on the epithelialbasement membrane side (luminal) of a sheet of UBM attached to UBM,proliferated, differentiated and formed a confluent monolayer followingthree days' growth.

Application 3:

It is contemplated that the tissue graft composition of the presentinvention can be used to induce repair or replacement of tissue in vivo,including connective tissues, such as ligaments, tendons, cartilage,bone, joints, and muscle, epithelial tissues, such as urinary bladder,and other tissues of the urogenital tract, stomach, esophagus, and othertissues of the gastrointestinal tract, liver, nervous tissue, tissues ofthe head and neck, skin, and other tissues using the same proceduresdescribed in U.S. Pat. Nos. 4,902,508; 4,956,178; 5,281,422; 5,352,463;5,554,389; 5,275,826; 4,902,508; 5,372,821; 5,445,833; 5,516,533;5,573,784; 5,641,518; 5,695,998; 5,711,969; 5,755,791; 5,762,966; and5,885,619, the disclosures of which are incorporated herein byreference. The tissue graft composition of the invention can also beused with synthetic or non-synthetic polymers for restoration oftissues.

What is claimed is: 1-17. (canceled)
 18. A method for extracting cellsfrom a tissue, comprising: soaking said tissue in a hypertonic solutionfor a period of time sufficient to remove substantially all cellularmaterial.